Detection of the presence and the determination of the amount of a specific protein using various techniques utilizing antibodies specific to the target protein are one of the most important tools in biomedical research, medical diagnosis and detection of biological threat agents. These techniques (such as, for example, ELISA) take advantage of the exquisite specificity of antigen binding by the antibodies and constitute the current gold-standard of specific protein detection. While extremely useful, these existing techniques have some limitations. One important limitation is relatively long time required to perform the assay. This limitation is especially important in situations where the answers need to be known immediately such as, for example, in determination of biomarkers important for emergency room care decisions. Another important limitation is a relative technical complexity of these assays which limits their point-of-care applicability and their adaptation and tailoring to the needs of the intended end-users and their settings. The major goal of this project will be to develop a new antibody-based protein detection methodology (molecular pincers), which will retain all advantages of classical antibody-based assays while reducing the time, complexity, and the cost of the assay. This new methodology is an extension of nucleic-acid-based "molecular beacon" methodologies previously developed by us. This new assay format is homogeneous requiring no complicated sample manipulations. A sample needs only to be added to the assay mixture and after a short incubation an easy to read optical signal (fluorescence) reports the presence of the target molecule. While the molecular pincers will be applicable to any situation involving antibody-based detection of antigens, their unique properties and characteristics make them especially applicable in situations requiring rapid results. In Phase I of this project we will use cardiac troponin, an excellent example of a cardiac biomarker requiring quick determination in emergency room in patients with chest pains, as a model target protein to test the concept of molecular pincers. While all of the proposed research in Phase I will involve cardiac troponin as a model, the lessons related to molecular pincer design will be generally applicable to other systems. Following successful completion of Phase I, in Phase II molecular pincers to an extensive panel of proteins will be developed, tested and validated. A priority in Phase II will be given to target proteins with an established need for their rapid determination. Additional goal of Phase II will be to develop signal amplification procedures compatible with molecular pincer design to extend sensitivity of the assay. In Phase I of this proposal we will test and validate two designs of molecular pincers: Aim #1: To develop and test molecular pincer assay for detecting troponin based on two antibodies recognizing two nonoverlaping epitopes of troponin. Aim #2: To develop and test single antibody-based molecular pincer assay for detecting troponin based on competition between the protein and the epitope-containing synthetic peptide. Successful completion of the above aims will validate the practical feasibility of molecular pincer design. We expect that molecular pincers will find very broad applications as a diagnostic tool allowing rapid and straightforward tests for biomarkers of human disease and as a research tool replacing, when possible, ELISA tests with a much simpler to perform homogeneous assay. [unreadable] [unreadable] [unreadable]